Endotoxin binding by lactic acid bacteria and bifidobacteria

ABSTRACT

The invention relates to the use of at least one strain of lactic acid bacteria and/or bifidobacteria having hydrophobic surface properties, for the preparation of a composition intended for the prevention or the treatment of endotoxin mediated and/or associated disorders and human or pet food compositions prepared thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/470,807, filed Feb. 19, 2004, now U.S. Pat. No. 7,550,285, which is anational stage designation of International Application No.PCT/EP02/01163, filed Feb. 1, 2002, which claims priority to EuropeanApplication No. 0120043.2, filed Feb. 6, 2001, the entire contents ofwhich are expressly incorporated herein by reference thereto.

The present invention relates to the use of lactic acid bacteria and/orbifidobacteria that are hydrophobic for the preparation of a foodcomposition intended for the prevention or the treatment of endotoxinmediated and/or associated disorders. The invention also relates tocomposition prepared thereof.

BACKGROUND OF THE INVENTION

Sepsis-inducing toxins have been found associated with pathogenicbacteria, viruses, plants and venom. Among the well-described bacterialtoxins are the endotoxins or lipopolysaccharides (LPS) of thegram-negative bacteria. These molecules are glycolipids that areubiquitous in the outer membrane of all gram-negative bacteria, whichare believed responsible for gram-negative sepsis. This type of sepsisis an extremely common condition and is often fatal.

A number of approaches for treating sepsis have been attempted. Theseinclude use of antibodies to LPS, use of antibodies to tumour necrosisfactor, use of a soluble TNF receptor, use of a soluble interleukin-1(IL-1) receptor, to name a few. While each approach has some efficacy,the overall results have been disappointing.

Others have attempted to design and study proteins which bindLPS/endotoxin, and illustrative reports of these attempts appear inRustici, A. et al. Science (1993) 259:361-364; Matsuzaki, K. et al.Biochemistry (1993) 32:11704-11710; Hoess, A. et al. EMBO J (1993)12:3351-3356; and Elsbach, P. et al. Current Opinion in Immunology(1993) 5:103-107. In fact, upon introduction of LPS into the blood, itmay bind to a protein termed lipopolysaccharide-binding protein (LBP).Inhibition of LBP, e.g., with an anti-LBP antibody, has been suggestedas therapeutically useful for treating endotoxin-mediated sepsis(International Patent Application No. PCT/US90/04250, filed Jul. 30,1990). Also, work from several laboratories has shown that plasmalipoproteins, particularly high-density lipoproteins (HDL), bind andneutralise LPS (Skarnes et al., 1968, J. Bacteriology 95:2031; Flegel etal., 1993, Infect. Immunol. 61(12):5140) and that these particles mayconstitute the LPS-neutralising activity in plasma.

Previous treatments for endotoxin mediated and/or associated diseaseshas been retrospective (i.e., after development of clinical illness) andhas been limited to chemotherapeutic intervention. Prevention measureswere not achieved with such treatments.

Thus, there is a need in the art for an effective agent for neutralisinggram-negative endotoxin (i.e., LPS), in order to prevent or alleviatesymptoms of sepsis and septic shock.

The hydrophobic lactic acid bacteria and bifidobacteria of the presentinvention provide additional compounds which are capable of bindingendotoxins and ameliorating/preventing its effects.

SUMMARY OF THE INVENTION

Thus, the present invention relates to the use of at least one strain oflactic acid bacteria and/or bifidobacteria that has hydrophobic surfaceproperties, for the preparation of a composition intended for theprevention or the treatment of endotoxin mediated and/or associateddisorders.

In fact, it has been surprisingly found that some lactic acid bacteriaand bifidobacteria, particularly those with hydrophobic surface, havethe ability to bind endotoxins. Thus, allowing their use as efficientagents for prevention of endotoxic shock and sepsis of gut origin,bacterial translocation, necrotising enterocolitis, inflammatory boweldisease, intestinal infections, chronic endotoxemia associated orpromoting catabolic and systemic inflammation.

Preferably, the hydrophobic lactic acid bacteria or bifidobacteria has apercent hydrophobicity (% H) of at least 80, and more preferably from 85to 100% H.

In a preferred embodiment, the strain is selected from the groupconsisting of Lactobacillus johnsonii, Lactobacillus reuterii,Lactobacillus paracasei, Lactobacillus animalis, Lactobacillus ruminis,Lactobacillus acidophilus, Lactobacillus rahmnosus, Lactobacillusfermentum, Lactobacillus delbrueckii subs. lactis, Bifidobacterium spp.,Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacteriumpseudolongum, Bifidobacterium infantis, Bifidobacterium adolescentis.

In another aspect, the invention relates to an isolated strain of lacticacid bacteria or bifidobacteria having hydrophobic surface properties,that has been selected for its ability to bind endotoxins orco-aggregate with gram-negative bacteria.

In a further aspect, the invention provides a human or pet foodcomposition for preventing or treating endotoxin mediated and/orassociated disorders, which contains at least a strain of lactic acidbacteria and/or bifidobacteria having the above traits, associated withan ingestible support or a pharmaceutical matrix.

This composition presents the advantage to decrease small bowelbacterial overgrowth and diminish the endotoxin leakage from the gut tothe internal milieu, a frequent disorder found in pets that may causeepisodes of diarrhoea, malnutrition and intestinal and systemicinflammation, for example.

In a last aspect, the invention relates to a method of preventing ortreating endotoxin mediated and/or associated disorders, comprising thestep of feeding a human or animal a composition which contains at leasta strain of lactic acid bacteria and/or bifidobacteria that hashydrophobic surface properties, associated with an ingestible support ora pharmaceutical matrix

DETAILED DESCRIPTION OF THE INVENTION

Within the following description, “NCC” designates Nestlé CultureCollection (Nestlé Research Centre, Vers-chez-les-Blanc, Lausanne,Switzerland).

With respect to the first object of the present invention, the use of atleast one strain of lactic acid bacteria and/or bifidobacteria that hashydrophobic surface properties, for the preparation of a compositionintended for the prevention or the treatment of endotoxin mediatedand/or associated disorders, is concerned.

In fact, it has been surprisingly found that some lactic acid bacteriaand bifidobacteria, particularly those with hydrophobic surface, havethe ability to bind endotoxins.

Indeed, the bacterial strain according to the invention has the abilityto bind endotoxins on the hydrophobic cell wall and therefore scavengethis proinflammatory product of gram negative bacteria that otherwisemay translocate from the lumen of the gut into the blood and therebytrigger inflammatory reactions and, in very serious cases, endotoxicshock.

The lactic acid bacteria or bifidobacteria according to the inventionhave been selected among strains suitable for animal consumption, withregard to their percent hydrophobicity (% H), as described in A. G.Zavaglia et al., Journal of Food protection, Vol. 61, No. 7, 1998, p.865-873.

Preferably, the bacterial strain according to the invention has a % H ofat least 80, and more preferably from 85 to 100% H.

The determination of surface hydrophobicity was done by using the MATHmethod as previously described (Pérez, P. F. et al., 1998, Appl.Environ. Microbiol. 64: 21-26). Briefly, 2 ml of bacterial suspension(around 10⁸ CFU/ml, PBS) were extracted with 0.4 ml of xylene byvortexing them during 120 s. The phases were allowed to separe bydecantation and A₆₀₀ of the aqueous phase was measured. Cell surfacehydrophobicity (% H) was calculated with the formula H %=[(A₀−A)/A₀]×100where A₀ and A are absorbances before and after extraction with xylenerespectively.

In a preferred embodiment the bacterial strain may be selected from thegroup consisting of Lactobacillus johnsonii, Lactobacillus reuterii,Lactobacillus paracasei, Lactobacillus animalis, Lactobacillus ruminis,Lactobacillus acidophilus, Lactobacillus rahmnosus, Lactobacillusfermentum, Lactobacillus delbrueckii subs. lactis Bifidobacterium spp.,Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacteriumpseudolongum, Bifidobacterium infantis, Bifidobacterium adolescentis.

In a most preferred embodiment the strain may be Lactobacillusacidophilus NCC 2463 (CNCM-I 2623), Bifidobacterium bifidum NCC 189(previously CIDCA 536, CNCM-I-2333), Bifidobacterium bifidum NCC 235(previously CIDCA 533, CNCM-I-2335), Bifidobacterium adolescentis NCC251 (CNCM 1-2168), Bifidobacterium lactis (ATCC27536), for example.

Among the various strains selected in accordance with the presentinvention, the following strains were deposited by way of example underthe Budapest Treaty at the Collection Nationale de Cultures deMicroorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724Paris Cedex 15, France, Lactobacillus acidophilus NCC 2463 on the 2 Feb.2001 under the reference CNCM I-2623, and Bifidobacterium bifidum NCC189 and NCC 235 on the 12 Oct. 1999 under the references CNCM-I-2333 andCNCM 1-2335, respectively. Bifidobacterium adolescentis NCC 251 wasdeposited on the 15 Mar. 1999 under the reference CNCM I-2168.

The strain of Bifidobacterium lactis (Bb12) (ATCC27536) is provided byHansen (Chr. Hansen A/S, 10-12 Boege Alle, P.O. Box 407, DK-2970Hoersholm, Danemark). It has a hydrophobicity of 89% H.

The bacterial strain according to the present invention may be used forthe preparation of compositions intended for improving human or animalhealth, particularly for the prevention or treatment of disordersrelated to endotoxins in humans and pets. The bacterial strain may beused as efficient agent for prevention of endotoxic shock and sepsis ofgut origin, bacterial translocation, necrotising enterocolitis,inflammatory bowel disease, intestinal infections, and chronicendotoxemia associated or promoting catabolic and systemic inflammation,for example.

The bacterial strain according to the invention may be used in itsviable or inactivated form.

In a preferred embodiment the lactic bacteria strain is used in thepresence of its fermented growth medium. The said medium can be eithersterilised alone or with a food, extruded or spray-dried, chilled orshelf stable, for example.

The bacterial strain may be used so that the amount available for theindividual may correspond to about 10³-10¹⁴ cfu per day. This amountdepends on the individual weight, and it is preferably of about 10⁹-10¹²cfu/day for humans and 10⁷-10¹⁰ cfu/day for pets.

According to another aspect, the present invention relates to anisolated strain of lactic acid bacteria or bifidobacteria havinghydrophobic surface properties and having the ability to bind endotoxinsor co-aggregate with gram-negative bacteria.

The ability of the bacterial strain according to the present inventionto bind endotoxins can be measured easily using FITC-labelledendotoxins, measuring the association of radiolabelled endotoxin tobacterial cells, in this case the molecular structure of the endotoxinis unaltered as compared with the possible modifications that may sufferthe molecule after conjugation with the FITC (see examples).

Preferably, the capacity of the bacterial strain to remove endotoxinfrom a solution that mimics the quantity of endotoxins found in thehuman intestine is measured. For example, levels of endotoxins wereexamined with the microassay for the detection of 2-keto-3-deoxyoctonategroup in the lipopolysaccharide of gram-negative bacteria (Karkhanis YD, et al., Analytical Chemistry (1978) 85: 595-601). Bacteria removingmore that 30% of the endotxin content from these solutions werepreferably selected. The non-hydrophobic bacteria tested in this assaywere unable to modify the initial content of endotoxin (see examples).

Such bacterial strain may be used as described above, and particularlyas efficient agent for prevention of endotoxic shock and sepsis of gutorigin, bacterial translocation, necrotising enterocolitis, inflammatorybowel disease, intestinal infections, chronic endotoxemia associated orpromoting catabolic and systemic inflammation, for example.

According to a further aspect, the present invention relates to a humanor pet food composition containing at least an isolated strain of lacticacid bacteria and/or bifidobacteria, said strain having the abovetraits, associated with an ingestible support or a pharmaceuticalmatrix.

The strain may be selected as described above.

Preferably, the lactic acid bacteria or bifidobacteria may beadministered as a supplement to the normal diet or as a component of anutritionally complete human or pet food.

The human or pet food composition may comprise at least the lactic acidbacteria and/or bifidobacteria strain, as described above, so that theamount available for the individual may correspond to about 10³-10¹⁴ cfuper day. This amount depends on the individual weight, and it ispreferably of about 10⁹-10¹² cfu/day for humans and 10⁷-10¹⁰ cfu/day forpets.

The human food may be in the form of a nutritional formula, an infantformula, milk-based products, dairy products, cereal-based products, forexample. To prepare such a food product or composition, the bacterialstrain as described above can be incorporated into a food, such ascereal powder, milk powder, a yoghurt, during its manufacture, forexample.

In one embodiment, a nutritional formula comprising a source of proteinand at least one bacterial strain according to the invention can beprepared. Dietary proteins are preferably used as a source of protein.The dietary proteins may be any suitable dietary protein; for exampleanimal proteins (such as milk proteins, meat proteins and egg proteins),vegetable proteins (such as soy, wheat, rice or pea proteins), mixturesof free amino acids, or combination thereof Milk proteins such ascasein, whey proteins and soy proteins are particularly preferred. Thecomposition may also contain a source of carbohydrates and a source offat.

If the nutritional formula includes a fat source, the fat sourcepreferably provides about 5% to about 55% of the energy of thenutritional formula; for example about 20% to about 50% of the energy.The lipids making up the fat source may be any suitable fat or fatmixtures. Vegetable fats are particularly suitable; for example soy oil,palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canolaoil, lecithin, and the like. Animal fats such as milk fats may also beadded if desired.

If the nutritional formula includes a carbohydrate source, thecarbohydrate source preferably provides about 40% to about 80% of theenergy of the nutritional formula. Any suitable carbohydrates may beused, for example sucrose, lactose, glucose, fructose, corn syrupsolids, and maltodextrins, and mixtures thereof.

Dietary fibre may also be added if desired. Numerous types of dietaryfibre are available. Suitable sources of dietary fibre, among others,may include soy, pea, oat, pectin, guar gum, and gum arabic. If used,the dietary fibre preferably comprises up to about 5% of the energy ofthe nutritional formula. Suitable vitamins and minerals may be includedin the nutritional formula in the usual manner to meet the appropriateguidelines. One or more food grade emulsifiers may be incorporated intothe nutritional formula if desired; for example diacetyl tartaric acidesters of mono-diglycerides, lecithin and mono- and di-glycerides.Similarly suitable salts and stabilisers may be included.

The nutritional formula is preferably enterally administrable; forexample in the form of a powder, a liquid concentrate, or aready-to-drink beverage.

The nutritional formula may be prepared in any suitable manner. Forexample, the nutritional formula may be prepared by blending togetherthe source of dietary protein, the carbohydrate source, and the fatsource in appropriate proportions. If used, the emulsifiers may beincluded in the blend. The vitamins and minerals may be added at thispoint but are usually added later to avoid thermal degradation. Anylipophilic vitamins, emulsifiers and the like may be dissolved into thefat source prior to blending. Water, preferably water that has beensubjected to reverse osmosis, may then be mixed in to form a liquidmixture. The temperature of the water is conveniently about 50° C. toabout 80° C. to aid dispersal of the ingredients. Commercially availableliquefiers may be used to form the liquid mixture. The liquid mixture isthen homogenised; for example in two stages.

The liquid mixture may then be thermally treated to reduce bacterialloads. For example, the liquid mixture may be rapidly heated to atemperature in the range of about 80° C. to about 150° C. for about 5seconds to about 5 minutes. This may be carried out by steam injection,autoclave or by heat exchanger; for example a plate heat exchanger. Theliquid mixture may then be cooled to about 60° C. to about 85° C.; forexample by flash cooling. The liquid mixture may then be againhomogenised; for example in two stages at about 7 MPa to about 40 MPa inthe first stage and about 2 MPa to about 14 MPa in the second stage. Thehomogenised mixture may then be further cooled to add any heat sensitivecomponents; such as vitamins and minerals. The pH and solids content ofthe homogenised mixture is conveniently standardised at this point.

If it is desired to produce a powdered nutritional formula, thehomogenised mixture is transferred to a suitable drying apparatus suchas a spray drier or freeze drier and converted to powder. The powdershould have a moisture content of less than about 5% by weight.

If it is desired to produce a liquid formula, the homogenised mixture ispreferably aseptically filled into suitable containers. Aseptic fillingof the containers may be carried out by pre-heating the homogenisedmixture (for example to about 75 to 85° C.) and then injecting steaminto the homogenised mixture to raise the temperature to about 140 to160° C.; for example at about 150° C. The homogenised mixture may thenbe cooled, for example by flash cooling, to a temperature of about 75 to85° C. The homogenised mixture may then be homogenised, further cooledto about room temperature and filled into containers. Suitable apparatusfor carrying out aseptic filling of this nature is commerciallyavailable. The liquid formula may be in the form of a ready to feedformula having a solids content of about 10 to about 14% by weight ormay be in the form of a concentrate; usually of solids content of about20 to about 26% by weight. Flavours may be added to the liquid formulasso that the formulas are provided in the form of convenient,flavoursome, ready-to-drink beverages.

In an another embodiment, a usual food product may be enriched with thebacterial strain according to the present invention. For example, afermented milk, yoghurt, a fresh cheese, a renneted milk, aconfectionery bar, breakfast cereal flakes or bars, drinks, milkpowders, soy-based products, non-milk fermented products or nutritionalsupplements for clinical nutrition.

In a further embodiment, a nutritionally complete pet food compositioncan be prepared. It may be in powdered, dried form, semi-moist or a wet,chilled or shelf stable pet food product. It can also be dietarysupplements for pets or pharmaceutical compositions. These pet foods maybe produced as is conventional. Apart from the bacteria strain, thesepet foods may include any one or more of a starch source, a proteinsource and lipid source.

Suitable starch sources are, for example, grains and legumes such ascorn, rice, wheat, barley, oats, soy, and mixtures of these. Suitableprotein sources may be selected from any suitable animal or vegetableprotein source; for example meat and meal, poultry meal, fish meal, soyprotein concentrates, milk proteins, gluten, and the like. For elderlyanimals, it is preferred for the protein source to contain a highquality protein. Suitable lipid sources include meats, animal fats andvegetable fats. Further, various other ingredients, for example, sugar,salt, spices, seasonings, vitamins, minerals, flavouring agents, fatsand the like may also be incorporated into the pet food as desired.

For dried pet foods a suitable process is extrusion cooking, althoughbaking and other suitable processes may be used. When extrusion cooked,the dried pet food is usually provided in the form of a kibble. If aprebiotic is used, the prebiotic may be admixed with the otheringredients of the dried pet food prior to processing. A suitableprocess is described in European patent application No 0850569; thedisclosure of which is incorporated by reference. If a probioticmicro-organism is used, the organism is best coated onto or filled intothe dried pet food. A suitable process is described in European patentapplication No 0862863; the disclosure of which is incorporated byreference.

For wet foods, the processes described in U.S. Pat. Nos. 4,781,939 and5,132,137 may be used to produce simulated meat products. Thedisclosures of these patents are incorporated by reference. Otherprocedures for producing chunk type products may also be used; forexample cooking in a steam oven. Alternatively, loaf type products maybe produced by emulsifying a suitable meat material to produce a meatemulsion, adding a suitable gelling agent, and heating the meat emulsionprior to filling into cans or other containers.

The amount of prebiotic in the pet food is preferably about 20% byweight; especially about 10% by weight. For example, the prebiotic maycomprise about 0.1% to about 5% by weight of the pet food. For pet foodswhich use chicory as the prebiotic, the chicory may be included tocomprise about 0.5% to about 10% by weight of the feed mixture; morepreferably about 1% to about 5% by weight.

If a probiotic microorganism is used, the pet food preferably containsabout 10⁴ to about 10¹⁰ cells of the probiotic microorganism per gram ofthe pet food; more preferably about 10⁶ to about 10⁸ cells of theprobiotic microorganism per gram. The pet food may contain about 0.5% toabout 20% by weight of the mixture of the probiotic microorganism;preferably about 1% to about 6% by weight; for example about 3% to about6% by weight.

The pet foods may contain other active agents such as long chain fattyacids. Suitable long chain fatty acids include alpha-linoleic acid,gamma linoleic acid, linoleic acid, eicosapentanoic acid, anddocosahexanoic acid. Fish oils are a suitable source of eicosapentanoicacids and docosahexanoic acid. Borage oil, blackcurrent seed oil andevening primrose oil are suitable sources of gamma linoleic acid.Safflower oils, sunflower oils, corn oils and soybean oils are suitablesources of linoleic acid. If necessary, the pet foods are supplementedwith minerals and vitamins so that they are nutritionally complete.

Further, if desired, the bacteria strain may be encapsulated; forexample in a sugar matrix, fat matrix or polysaccharide matrix.

The amount of the pet food to be consumed by the pet to obtain abeneficial effect will depend upon the size or the pet, the type of pet,and age of the pet. However an amount of the pet food to provide a dailyamount of about 10³-10¹⁴ cfu of at least one lactic acid bacteria orbifidobacteria strain would usually be adequate. Preferably about10⁹-10¹⁰ cfu/day for dogs or 10⁷-10¹⁰ cfu/day for cats are administered,for example.

The composition according to the invention is particularly intended forthe prophylaxis or the treatment of infections related to gram negativebacteria, endotoxin producing bacteria such as Helicobacter spp,Samonella spp, and also to small intestinal bacterial overgrowth (SIBO)all of which may clinically manifest with diarrhoea, intestinal orsystemic inflammatory conditions, or catabolism and malnutrition.

According to a last aspect, the invention provides a method ofpreventing or treating endotoxin mediated and/or associated disorders,comprising the step of feeding a human or animal a composition whichcontains at least a strain of lactic acid bacteria and/or bifidobacteriathat has hydrophobic surface properties, associated with an ingestiblesupport or a pharmaceutical matrix.

This method may be particularly intended for the prophylaxis or thetreatment of infections related to gram negative bacteria, endotoxinproducing bacteria such as Helicobacter spp, Samonella spp, and also tosmall intestinal bacterial overgrowth (SIBO) all of which may clinicallymanifest with diarrhoea, intestinal or systemic inflammatory conditions,or catabolism and malnutrition.

The following examples are given by way of illustration only and in noway should be construed as limiting the subject matter of the presentapplication. All percentages are given by weight unless otherwiseindicated. The examples are preceded by a brief description of thefigures.

FIGURES

FIG. 1: Histogram plots for Bifidobacterium bifidum strain NCC 189(previously CIDCA 536, CNCM I-2333) showing binding of FITC-LPS. A:control without FITC-LPS. B: incubation with 50 μl/ml FITC-LPS. At least16000 events were analysed.

FIG. 2: Effect of albumin on FITC-LPS binding by lactic acid bacteriaand bifidobacteria. A: Bifidobacterium bifidum strain NCC 200(previously CIDCA 538, CNCM I-2334). B: Lactobacillus acidophilus strainNCC 2463 (CNCM I-2623). C: Bifidobacterium bifidum strain NCC 189(previously CIDCA 536, CNCM I-2333).

FIG. 3: Kinetics of growth and FITC-LPS binding for Bifidobacteriumbifidum strain NCC 189 (previously CIDCA 536, CNCM 1-2333). Values aremeans from two independent experiments.

FIG. 4: Binding of LPS by Bifidobacterium strain, detected by themicroassay to determine 2-keto-3-deoxyoctonato (KDO) inlipopolysaccharide. The initial concentration of the solution was 100μg/ml. The bars show the final concentration after incubation of thesolution with hydrophobic bifidobacterial strains NCC 189 and NCC 251(CNCM I-2168) compared with a non-hydrophobic strain of bifidobacteria(NCC 200 (CNCM I-2334)).

FIG. 5: Proinflammatory activity (IL-8 (ng/mL)) of LPS solutionpreincubated with hydrophobic bacteria on HT-29 human epithelial cells.

Control samples of DMEM, Human milk (HM) 2%, LPS (2.5 μg/ml) alone,Bifidobacterium bifidum strain NCC 189 (1.5e8 cfu/ml) alone were testedfor background stimulatory activity. Test solutions of LPS+human milk(source of sCD14) were compared with solution containing LPS 2.5μg/ml+HM 2% preincubated with Bifidobacterium bifidum strain NCC189(1.5e8 cfu/ml).

EXAMPLES Example 1 Selection of Hydrophobic Lactic Acid Bacteria StrainAccording to the Invention

The selection of hydrophobic bacteria was initially based in the % ofpartition of bacterial cells between an organic (hydrophobic) and anaqueous phase. Determination of surface hydrophobicity was done by usingthe MATH method as previously described (Pérez, P. F. et al., 1998,Appl. Environ. Microbiol. 64: 21-26). Briefly, 2 ml of bacterialsuspension (around 10⁸ CFU/ml, PBS) were extracted with 0.4 ml of xyleneby vortexing them during 120 s. The phases were allowed to separe bydecantation and A₆₀₀ of the aqueous phase was measured. Cell surfacehydrophobicity (% H) was calculated with the formula H %=[(A₀−A)/A₀]×100where A₀ and A are absorbances before and after extraction with xylenerespectively.

Subsequently lactic acid bacteria or bifidobacteria were selected bytheir capacity to remove endotoxin from a solution that mimics thequantity of endotoxin found in the human intestine. Levels of endotoxinwere examined with the microassay for the detection of2-keto-3-deoxyoctonate group in the lipopolisaccharide of gram-negativebacteria (Karkhanis Y D, et al., Analytical Chemistry (1978)85:595-601).

Bacteria removing more that 30% of the endotoxin content from thesesolutions were selected. The non-hydrophobic bacteria tested in thisassay were unable to modify the initial content of endotoxin.

Example 2 In-Vitro Effect of Lactic Acid Bacteria as Endotoxin Scavenger

The interaction between LPS from Escherichia coli and lactic acidbacteria bearing different surface properties was studied havingselected bacteria with more of 80% H and some non-hydrophobic “negative”controls the interaction with fluorescent labelled endotoxin wasperformed with flow cytometry.

Materials and Methods

Bacterial Strains and Growth Conditions

Strain Lactobacillus acidophilus NCC 2463 (CNCM I-2623) was from Nesteccollection (Lausanne, Switzerland). Strains Bifidobacterium bifidum NCC189 (previously CIDCA 536, I-2333) and Bifidobacterium bifidum NCC 200(previously CIDCA 538, (CNCM I-2334)) were from the collection of theCentro de Investigacion y Desarrollo en Criotecnologia de Alimentos (Laplata, Argentina). Frozen suspensions (−80° C.) preserved with 10%(vol./vol.) glycerol were reactivated once in MRS broth beforeexperiments. All cultures were conducted at 37° C. in anaerobicconditions (BBL GasPak Plus).

FITC-LPS Binding

Lipopolysaccharide and FITC-labelled lypopolysaccharide were fromEscherichia coli serotype 0111:B4 (Skelly, R. R et al., 1979, Infect.Immun. 23: 287-293) and were purchased by Sigma. Stock solutionscontaining 1000 μl/ml were prepared in distilled water and dilutedappropriately.

Bacterial cultures were washed three times with PBS and suspensions werestandardised to 10⁷ CFU/ml. 400 μl were mixed with different amounts ofFITC-LPS or LPS to obtain concentrations ranging from 0 to 50 μg/ml inthe reaction mixture. Incubations were performed at 4° C. or 37° C.during 30 minutes and then, cells were washed two times with PBS andfixed with paraformaldehyde 1% (vol./vol.) during 30 minutes at 4° C.Flow cytometric analysis was done using a blue-green excitation light(FACScan™).

Surface Hydrophobicity

Determination of surface hydrophobicity was done by using the MATHmethod as previously described (Pérez, P. F. et al., 1998, Appl.Environ. Microbiol. 64: 21-26). Briefly, 2 ml of bacterial suspension(around 10⁸ CFU/ml, PBS) were extracted with 0.4 ml of xylene byvortexing them during 120 s. The phases were allowed to separe bydecantation and A₆₀₀ of the aqueous phase was measured. Cell surfacehydrophobicity (% H) was calculated with the formula H %=[(A₀−A)/A₀]×100where A₀ and A are absorbances before and after extraction with xylenerespectively.

Results

Surface hydrophobicities of strains under study are shown in Table 1.Values were 93 and 96% for strains Lactobacillus acidophilus NCC 2463(CNCM I-2623) and Bifidobacterium bifidum NCC 189 (CNCM I-2333)respectively whereas strain Bifidobacterium infantis NCC 200 (CNCMI-2334) was not hydrophobic and showed values around 3%.

TABLE 1 Hydrophobicities of strains under study. Values are means of atleast 3 determinations. Strain Hydrophobicity % NCC 2463 (CNCM I-2623)93 ± 3 NCC 189(CNCM I-2333) 96 ± 3 NCC 200 (CNCM I-2334)  3 ± 2

Incubation of strain NCC 189 with 50 μg/ml of FITC-labelled LPS clearlyshifts bacterial population into a fluorescent zone. Events situated inthe right side of the graph (marker M1) represents 90% of the gatedpopulation (FIG. 1B). For suspensions incubated without FITC-LPS, only2% of the events are situated in different binding capability.

Table 2 (below) shows that FITC-LPS binding is dose dependent. 5 and 15%of FITC (+) cells were found for strains NCC200. On the other hand,strain NCC 189 shows around 95% of FITC (+) cells. Saturation occursaround 10 μg/ml of FITC-LPS. Addition of albumin, a lipid binder,clearly reduces FITC (+) ratio in all strains under study (FIG. 2).However, strain CIDCA 536 shows around 30% of FITC (+) cells in thepresence of 0.4% albumin and 50 μg/ml of FITC-LPS (FIG. 2C). Furthermorefor this strain, 10% of FITC (+) cells were found with albuminconcentrations as high as 3%. Divalent cations (Ca2+0.9 mM and Mg2+0.5mM) did not modified binding (data not shown).

TABLE 2 Percent of FITC (+) bacteria at different concentrations ofFITC-LPS. At least 30000 events were analysed. FITC-LPS Strain (μg/ml)NCC 189 NCC 200 0 0.1 0.2 1 88.1 0.7 10 92.1 5.2 20 92.8 5.9 30 93.9 3.740 94.7 4.6 50 94.8 4.6

Binding of FITC-LPS for Bifidobacterium bifidum strain NCC 189(previously CIDCA 536, CNCM I-2333) was strongly dependent on growthphase (FIG. 3). Bacteria harvested in stationary phase shows a highratio of FITC (+) cells. These values fall dramatically during lag phaseand were progressively restored during growth. No differences betweenbinding at 4° C. and 37° C. were found.

In summary, highly hydrophobic strains show FITC (+) cells that reacharound 95% for strain NCC 189. Non hydrophobic strain NCC 200 neverreaches values higher than 5%. These results show that binding of LPScorrelates with surface hydrophobicity.

These results clearly indicated that hydrophobic bacteria bindFITC-labelled endotoxin and thereby become fluorescent. At aconcentration of 10 μg/ml of endotoxin more than 90% of the hydrophobiccell cultures become fluorescent as detected with flow cytometry; incontrast, less than 10% of non-hydrophobic bacterial cells becamepositive under similar endotoxin concentrations.

When hydrophobic cultures were introduced in media containing endotoxinat concentration ranging between 30 and 90 μg/ml, bacterial cultures atconcentration of 10⁷ to 10⁸ bacterial per ml removed at least 20% of thepresent endotoxin. This data was confirmed using radiolabelledendotoxin.

Example 3 Experiments on Human Immunocompetent Cells

The abrogation of the pro-inflammatory reaction in human immunocompetentcells induced by endotoxin by the presence in the culture system ofhydrophobic strains in comparison with non-hydrophobic bacteria wasstudied.

Hydrophobic Bifidobacterium bifidum NCC 189 (CNCM I-2333) andnon-hydrophobic Bifidobacterium infantis NCC 200 (CNCM I-2334) wereincubated in a solution containing a defined quantity of endotoxin asdetermined by the KDO method (see example 1) (FIG. 4).

Endotoxin presence after incubation with bacteria suspension was clearlydiminished by the hydrophobic strain whereas no changes were observedfor the non-hydrophobic strain.

Example 4 Functional Studies for Hydrophobic Bacteria

Materials and Methods

Washed bacteria were resuspended in DMEM-high glucose (AMIMED) and werepreincubated with LPS of Escherichia coli O111: B4 (Sigma) at a finalconcentration of 2.5 μg/ml. After centrifugation 200 μl of supernatantor resuspended bacterial pellet were used to stimulate HT-29 epithelialcells.

After 20-h culture at 37° C. the HT-29 cell culture supernatant wasexamined for the presence of IL-8 using an ELISA technique.

Results.

The results are presented in FIG. 5. Proinflammatory activity of LPS issignificantly reduced when an LPS solution is preincubated withhydrophobic bacteria. After centrifugation the supernatant added to ahuman epithelial cell culture (in the presence of human milk since LPSstimulation is dependent on sCD14 presence) is significantly reducedwith respect to the supernatant that has not been preincubated with thehydrophobic bacteria.

Example 5 Infant Formula

To obtain an infant formula we prepare the following mixture containingfor 100 ml of formula: 0.5 to 5%, preferably 2% of peptides, 0.2 to 10%,preferably 4% of fat, 1 to 25%, preferably 8% of non-levan carbohydrates(including lactose 65%, maltodextrin 20%, starch 15%), and at least 10⁶cfu/ml of the following strains: Lactobacillus acidophilus NCC 2463(CNCM I-2623), Bifidobacterium bifidum NCC189 (CNCM I-2333),Bifidobacterium bifidum CNCM I-2335, Bidifobacterium adolescentis CNCMI-2168, in combination with traces of vitamins and oligoelements to meetdaily requirements, and 0.01 to 2%, preferably 0.3%, of minerals, and 50to 90%, preferably 75% of water.

Example 6 Use of Hydrophobic Lactic Acid Bacteria According to theInvention in Dairy Products

One or more strain of Bifidobacterium bifidum (CNCM I-2333),Lactobacillus acidophillus NCC 2463 (CNCM I-2623), Bifidobacteriumbifidum NCC235 (CNCM I-2335) or Bifidobacterium adolescentis NCC251(CNCM I-2168), according to the present invention may be used for themanufacture of fermented yoghurt-like milk products.

To do this, 11 of a milk product containing 2.8% of fats andsupplemented with 2% of skimmed milk powder and 6% of sucrose isprepared, it is pasteurised at 96° C. for 30 minutes and its temperatureis then lowered to 42° C. Precultures of a non-thickening strain ofStreptococcus thermophilus and of a non-viscous strain Lactobacillusbulgaricus are reactivated in a sterile MSK culture medium containing10% of reconstituted milk powder and 0.1% of commercial yeast extract.

A preculture of one or more of the strain is also reactivated in amedium containing 10% of reconstituted milk powder and 0.1% ofcommercial yeast extract with 1% sucrose. The pasteurised milk productis then inoculated with 1% of each of these reactivated precultures andthis milk product is then allowed to ferment at 32° C. until the pHreaches a value of 4.5. Fermented milks yoghurt-like products areproduced in this way and stored at 4° C.

Example 7 Dry Dog Food

A feed mixture is made up of about 58% by weight corn, about 5.5% byweight of corn gluten, about 22% by weight of chicken meal, 2.5% driedchicory, fermented milk by strains of Lactobacillus acidophilus NCC 2463(CNCM-I 2623)) so that the corresponding amount for the dog is about10⁹-10¹⁰ cfu/day, and salts, vitamins and minerals making up theremainder.

The fed mixture is fed into a preconditioner and moistened. Themoistened feed is then fed into an extruder-cooker and gelatinised. Thegelatinised matrix leaving the extruder is forced through a die andextruded. The extrudate is cut into pieces suitable for feeding to dogs,dried at about 110° C. for about 20 minutes, and cooled to form pellets.

This dry dog food is able to improve pet health, and particularlyprevents disorders related to endotoxins in pets.

1. An isolated biologically pure strain selected from the groupconsisting of Lactobacillus acidophilus NCC 2463 (CNCM-I 2623),Bifidobacterium bifidum NCC 189 (CNCM-I-2333), Bifidobacterium bifidumNCC 235 (CNCM-1-2335) and Bifidobacterium adolescentis NCC 251(CNCM-I-2168) each having hydrophobic surface properties and an abilityto bind endotoxins or co-aggregate with Gram negative bacteria.
 2. Thestrain of claim 1, wherein the strain binds at least 30% of endotoxinfrom solutions where the concentrations are similar to an intestinalcontent of endotoxin.
 3. The strain of claim 1, wherein the strain has apercent hydrophobicity of at least 80.